Automated sample fractionation prior to mass spectrometric analysis

ABSTRACT

Systems, apparatus, methods, and kits are provided for automated mass spectrometric analysis of small volumes of liquid samples, such as biological samples. The systems, apparatus, and kits may be used in facilities where high throughput of samples, as well as reliable and repeatable assay results with little training of staff, are needed. Such facilities include hospital emergency wards.

CROSS REFERENCE TO RELATED APPLICATIONS SECTION

This application is a continuation of co-pending U.S. application Ser.No. 14/750,693, which claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/017,170, filed Jun. 25, 2014, entitled“AUTOMATED SAMPLE FRACTIONATION PRIOR TO MASS SPECTROMETRIC ANALYSIS,”the contents of which are hereby incorporated by reference in theirentirety.

FIELD

The subject matter described herein relates to systems, apparatus,methods, and kits related to mass spectrometric analysis of liquidsamples of small volume, such as mass spectrometric analysis of bodilyfluids in a clinical setting.

BACKGROUND

Detection of specific chemical species in liquid solutions can be acomplicated task in which an array of analytical equipment is used. Insome instances, utilizing a marker, such as luminescent or radioactivemarkers which identify the target species, or analyte, can lackprecision or sensitivity in some cases. Increasingly, mass spectrometry(MS) with an atmospheric pressure ionization (API) source is used bythose seeking to quantify one or more analyte in a complex liquidsolution. Mass spectrometric analysis requires that the sample beionized, that is to say that the species in the sample have a mass and anet charge, either positive or negative. The atmospheric pressureionization source converts charge-neutral analytes into ions in the gasphase that can be analyzed with mass spectrometry.

An atmospheric pressure ionization source can achieve ionization ofspecies in a liquid sample in various ways. Some of the most commontechniques are electrospray ionization (ESI) and atmospheric pressurechemical ionization (APCI). These two techniques have in commonatomizing the sample by expelling the sample liquid through a narrowtube while heating the tube. Droplets of the sample liquid evaporateinto the constituents of the sample, including the target chemicalspecies. As the evaporated constituents of the sample travel from thenarrow tube towards the mass spectrometer inlet, they travel through alarge electrical potential and become ionized.

Mass spectrometric analysis is useful in quantifying medications ortoxins in biological samples like blood, urine, or tissue extracts, aswell as monitoring pesticides or pollutants in food or water. Thesetypes of sample solutions may contain high concentrations of salts orbuffers, such as pH buffers, and these buffers make the ionization ofanalytes using electrospray ionization (ESI) or atmospheric pressurechemical ionization (APCI) highly inefficient. This effect is known asion suppression and is the direct result of excess of salt becomingionized instead of the analytes of interest. Currently, analysts usingmass spectrometry (MS) fractionate complex samples prior to MS analysis,effectively separating out much of the analytes of interest from thesalts or buffers prior to ionization. Fractionation is typically done byliquid chromatography (LC), most often high-pressure liquidchromatography (HPLC). Analysts use various chromatography media,solvents, additives and temperature to optimize the fractionation ofanalytes from interferents, such as salts and buffers. The directcoupling of high-pressure liquid chromatography (HPLC) fractionation tomass spectrometric (MS) analysis is very common and is known ashigh-pressure liquid chromatography-mass spectrometry (HPLC-MS).

Commercial systems for high-pressure liquid chromatography massspectrometry (HPLC-MS) can be large, complicated systems. Such systemscan include high-pressure liquid pumps, two or more solvent reservoirs,a solvent mixer to create the needed gradients in the solvent ratio, avalve or other sample introduction mechanism, a chromatography columnfor performing the fractionation, and a detector, which in the case ofHPLC-MS is a mass spectrometry detector. The complexity of commercialHPLC-MS systems can vary and such systems may have the ability toperform at different pressures, may include automated sampleintroduction, may include temperature controls, and may includeadditional optical detectors, for example those capable of measuringlight absorbance, fluorescence, or light scattering from a liquidsample.

When using a commercial high-pressure liquid chromatography-massspectrometry (HPLC-MS) system, the user should have some degree of skillor knowledge to obtain accurate and reliable results. The user optimizeseach analysis through multiple decisions, such as by selecting thecorrect chromatography media and solvents, though other variables can becontrolled, such as temperature, pressure, sample size, and detectioninstrument parameters. Optimization depends not only on the nature ofthe sample, but also on the target species, or analyte. Detection of ananalyte in blood can be very different from detecting the same analytein urine in that the optimal conditions for high-pressure liquidchromatography (HPLC) fractionation may not be the same. Thechromatography media that a skilled user selects may change according tothe contents of the sample, and the solvents the skilled user selectsneed to be compatible with the ionization process for mass spectrometricanalysis.

Commercial high-pressure liquid chromatography-mass spectrometry(HPLC-MS) systems may include two solvents (though one solvent ofvarying concentration can be used), a wash solvent and an elutionsolvent, being prepared for each analysis or batch of analysis. Askilled user prepares the solvents in concentrations appropriate for thesample and the selected chromatography media. It is typically the casethat the ratio between the wash and elution solvents changes over timeduring fractionation, and in commercial systems, a skilled user mayoversee this change in ratio between the solvents, which can also bethought of as a gradient in relative concentration in the solvents. Thereason for this gradient, or change in ratio, in the solvents is thatanalytes and contaminants have differential affinity for thechromatography media as compared to the elution solvent. In most cases,analytes are preferentially bound onto the chromatography media in thepresence of wash solvent, but as the relative amount of elution solventis increased, the analyte will eventually become unbound from thechromatography media and flow out of the system with the elutionsolvent. The skilled high-pressure liquid chromatography-massspectrometry (HPLC-MS) user understands the chemical properties of theanalytes in each sample and selects appropriate high-pressure liquidchromatography (HPLC) conditions, including the wash and elutionsolvents and their relative concentration during fractionation, toobtain sufficient distinction between the contaminants in a sample andthe desired analytes so that each can be detected.

The chromatographic columns used in commercial high-pressure liquidchromatography (HPLC) systems are quite large and designed for reuse.Since the media in the chromatographic columns are selected fordifferent types of fractionation, multiple columns will often beassociated with a commercial HPLC system. The size of the columns usedin commercial HPLC systems can require a large amount of sample, as wellas a large volume of solvents. Additionally, the intended long life-timeof the chromatographic columns means that they are used multiple times,and so the skilled user, or analyst, needs to be cognizant of what thecolumns were used for in the past in order to determine the veracity ofthe results he or she obtains.

SUMMARY

Methods, systems, and apparatus, including computer program products,are provided for analysis of small volumes of liquid samples, such asbiological samples, in systems using mass spectrometric analysis. Thesystems and apparatus include an analysis cartridge and a preloadedsolvent assembly.

In some aspects, provided herein are systems for determining theconcentration of an analyte of interest in a biological sample thatincludes a solvent assembly and a disposable cartridge. The solventassembly may include a first volume of a first solvent and a secondvolume of a second solvent, the solvent assembly may further include afirst outlet fluidly connected to the first volume and a second outletfluidly connected to the second volume. The disposable cartridge caninclude a first solvent introduction port and a second solventintroduction port, a sample introduction port, a chromatography region,and an outlet fluidly connected to the chromatography region. The firstsolvent introduction port may be configured to accept the first outletof the solvent assembly and to fluidly connect to the first volume, andthe second solvent introduction port may be configured to accept thesecond outlet of the solvent assembly and to fluidly connect to thesecond volume. The sample introduction port may be fluidly connected tothe first and second introduction ports. The chromatography region maybe fluidly connected to the sample introduction port.

The following features may be present in systems for determining theconcentration of an analyte of interest in a biological sample in anysuitable combination. In some embodiments, the system may furtherinclude a sample analysis portion that is operably connected to theoutlet of the disposable cartridge, in which the sample analysis portioncan include a mass spectrometric analysis system, in which the massspectrometric analysis system may produce a spectra for the analyte ofinterest, and may produce at least one of a plurality of isotopes of theanalyte of interest or a plurality of derivatized samples of the analyteof interest added to the biological sample in a single spectralmeasurement. The system may further include at least one processorhaving operational thereon computer instructions for at leastcontrolling operation of the system, to at least determine aconcentration of the analyte contained in the biological sample and toreport the at least one analyte concentration. In some embodiments, thesystem may include immobilized chromatography media in thechromatography region of the disposable cartridge. One or more filtersin the disposable cartridge may be included in the system, in which theone or more filters may be configured to remove precipitates and/orinsoluble material from the biological sample and yield a filteredbiological sample. In some such embodiments, the system may include afiltrate reservoir in the disposable cartridge, in which the filtratereservoir may have a metered volume and is configured to contain thefiltered biological sample. The cartridge may include one or moreinternal standards, in which the one or more internal standards areprovided in a dried form. The one or more internal standards maycorrespond to one or more predetermined assays. The immobilizedchromatography media may correspond to one or more predetermined assays.The solvent assembly and the disposable cartridge may fit together in aspecific orientation constrained by a key. In some embodiments, the keymay include an asymmetrical first outlet, an asymmetrical second outlet,both asymmetrical first and second outlets, or an asymmetrical joiningportion between the first and second outlets on the solvent assembly. Insome systems, the disposable cartridge may include a receiver structurethat complements the key and may be configured to allow the solventassembly and the disposable cartridge to fit together in the specificorientation constrained by the key. The receiver structure can includeat least one of the first solvent introduction port or the secondsolvent introduction port of the disposable cartridge. The first solventand the second solvent of the solvent assembly may be compatible withthe disposable cartridge to prepare the biological sample for an assayto determine the presence of the analyte of interest. The assay mayinclude at least one of a metabolic acidosis assay, an intentionaloverdose assay, an uncontrolled convulsions or seizures assay, a comaassay, or a cardiovascular assay. In some embodiments, the assay mayinclude one or more test panels. One or more codes indicating thecompatibility of the solvent assembly with the disposable cartridge toperform the assay to determine the presence of the analyte of interestmay be included in the system in some embodiments. The one or more codesmay include color coding, written codes, machine readable codes,electrical signaling codes, or any combination thereof. In some suchembodiments, the written codes may include alpha-numeric codes, symbols,or a combination of both. The machine readable codes may includesymbols, quick response (QR) codes, barcodes, or any combinationthereof. In some embodiments, the electrical signaling codes may includeradio frequency (RF) signals. One or more correct insertion structuresmay be included on the disposable cartridge, in which the one or morecorrect insertion features may be configured to orient the disposablecartridge correctly within the system. The one or more correct insertionstructures on the disposable cartridge may include a notched corner ofthe disposable cartridge. The first volume may be defined by a plungerassembly of a first syringe and the second volume may be defined by aplunger assembly of a second syringe. In some embodiments, a syringepump configured to independently dispense the first solvent and thesecond solvent. The system may include a first seal located at the firstoutlet of the solvent assembly and a second seal located at the secondoutlet of the solvent assembly, in which the first seal and the secondseal may prevent solvent from flowing out of the solvent assembly. Aseal piercing mechanism on each of the first and second solventintroduction ports can be included in the system, in which each sealpiercing mechanism is configured to break either the first seal or thesecond seal to allow solvent to flow into the disposable cartridge. Asolvent mixing chamber may be included in the disposable cartridge, inwhich the solvent mixing chamber may be configured to at least receivethe first and the second solvents from the solvent introduction port toform a mixed solvent, and may provide the mixed solvent to a reservoirthat is fluidly connected to the sample introduction port.

The above-noted aspects and features may be implemented in systems,kits, apparatus, methods, and/or articles depending on the desiredconfiguration. The details of one or more variations of the subjectmatter described herein are set forth in the accompanying drawings andthe description below. Features and advantages of the subject matterdescribed herein will be apparent from the description and drawings, andfrom the claims.

DESCRIPTION OF DRAWINGS

In the drawings,

FIG. 1 depicts a flow diagram of solution movement within an exemplaryanalysis system that includes a mass spectrometer;

FIG. 2 depicts an exemplary analysis system that includes inlet of twosolvents into a single-use, assay specific analysis cartridge;

FIG. 3 depicts a dual plunger assembly for wash and elution solvents foruse with a single-use, assay specific analysis cartridge;

FIG. 4 depicts top and bottom views of an assay specific analysiscartridge showing a solvent mixing chamber;

FIG. 5 depicts a detailed view of a tapered inlet port and a fluidicconnection to the mixing chamber shown in FIG. 4;

FIG. 6 depicts a solvent inlet port with a docked solvent syringe in anexemplary cartridge; and

FIG. 7 depicts an exemplary cartridge outlet port showing connection tostandard high-pressure liquid chromatography (HPLC) tubing and aninterface to a mass spectrometer.

Like labels are used to refer to same or similar items in the drawings.

DETAILED DESCRIPTION

In some example embodiments, there is provided apparatus, systems, kits,and methods that provide single use chromatography materials for samplepreparation for mass spectrometric (MS) analysis. Single-use, assayspecific analysis cartridges which fractionate a biological sample forMS analysis, as described herein, may allow for the use of small samplesize and may provide internal standards data analysis that can be usedwith single-use, disposable solvent-containing assembly. The single-use,assay specific cartridge in combination with the corresponding solventassembly, may remove the need for users to be highly skilled in mixingsolvents for chromatography; may allow for fewer errors, such as thosedue to reuse of fractionation media; may simplify the storage ofsolvents used in high-pressure liquid chromatography (HPLC); may reducethe amount of sample fluid needed; and/or may increase the speed andefficiency of obtaining high-pressure liquid chromatography-massspectrometry (HPLC-MS) results.

The systems, which include single-use assay specific cartridges thathave inlets for liquid samples, internal standards, fractionation media,and inlets for wash and elution solvents, as well as mass spectrometricanalysis instrumentation, can be used as shown in FIG. 1. A sample, suchas a biological sample, is collected, at 105, by a user. The biologicalsample may be blood, urine, or a homogenized tissue sample. The user mayplace, at 110, a small volume of the sample into a sample introductionport on the single-use cartridge. The sample inlet port may have one ormore filters to remove items from the sample that would complicatefractionation and items that are not needed for determination of thepresence of the target analyte. The one or more filters can remove itemssuch as blood platelets and cells. Within, or adjacent to, the sampleintroduction port are one or more types of internal standards, that isto say chemical species that are present in a known amount and thatshould yield known results when an elution solution containing thestandard is passed through a mass spectrometer. The internal standardsmay be provided in the single-use cartridge in a dried condition that isto say not in solution. After addition of the sample to the introductionport, the sample interacts, at 115, with the one or more internalstandards to bring them into solution. The filtered sample and theinternal standards are held in a filtrate reservoir beneath the sampleintroduction port and the one or more filters.

Once the sample is added to the introduction port on the single-use,assay specific cartridge, the user or the system seals, at 120, thesample introduction port that sits above the filtrate reservoir. Thismeans that any fluid that enters the filtrate reservoir from fluidconnections cannot exit through the sample introduction port. In someinstances, one-way valves, such as check valves or flap valves, mayguide the flow of fluid through the filtrate reservoir, so that fluidflows towards the chromatography portion of the single-use cartridge.

Without in any way affecting the scope of the claims, an advantage ofsome embodiments described herein is that a single-use solvent assemblyand a single-use assay specific cartridge for fractionation can enableverification that a particular solvent assembly is compatible with thesingle-use assay specific cartridge selected by the user. Written codes(e.g. numerical or letter codes), machine readable codes, quick response(QR) codes, bar codes, electronic codes (e.g., RF codes), color codes,physical compatibility (e.g. lock and key fitting that allows onlycompatible items to connect), or a combination thereof, can be used toidentify a solvent assembly as compatible with a single-use assayspecific cartridge. Once the user or system has confirmed that theintended solvent assembly is compatible with the cartridge that containsfiltered sample in the filtrate reservoir, the user or system introducessolvent into the cartridge through a solvent port, as in box 125. Theintroduction of solvent progresses the sample-solvent mixture towardsthe chromatography portion of the cartridge.

As time passes, the user or system supplies, at 130, the cartridge witha mixture of wash solvent and elution solvent in amounts chosen to becompatible with chromatography media in the cartridge, the sample type,and the target analyte so that impurities and the analyte of interestreach the mass spectrometry system at different times. The elutionsolution with the fractionated sample constituents and standards ispassed, at 135, to the mass spectrometric analysis portion of the systemto obtain reference data, as well as data regarding the analytes ofinterest, including the presence or absence of such analytes. Once themass spectrometric analysis portion of the system has completed datacollection, the data from the mass spectrometric analysis portion of thesystem is reported, at 140, to the user, clinician, or to another systemfor data analysis. For example, the data can be forwarded to a clinicianor a user, such as a caregiver, electronically, through a writtenreport, or both electronically and in a written report. Also, furthermanipulation of the data may be needed to provide a clinician or othercare giver with information that can be used to treat a patient, andanother system may perform such data manipulation if the cliniciancannot, for example due to time or lack of specific expertise.

FIG. 2 shows an example of an embodiment of a system for massspectrometric analysis that includes a solvent assembly 201, asingle-use assay specific cartridge 205, and a switch 225 that allowsfor toggling between a mass spectrometer 235 and a waste receptacle 260.The top portion 200A shows the system in a configuration suitable forflowing wash solvent through the cartridge 205. In this configuration,the user has inserted the sample through the sample introduction port215, and the sample has passed through one or more filters 220 into thefiltrate reservoir, along with one or more internal standards that arenow in solution. The one or more filters 220 remove cells, bloodplatelets, and/or other precipitates from the sample as it passes intothe filtrate reservoir. The solvent assembly 201 has sources for bothwash and elution solvents, assuming that they are distinct solvents.During the wash stage 200A, the wash solvent moves the sample and theone or more internal standards towards the chromatography portion 240 ofthe cartridge 205. The wash solvent flows over the chromatographyportion 240 for a predetermined amount of time, and this wash durationis controlled by the user or programmatically/automatically by thesystem. The switch 225 directs solvent to flow to a waste receptacle 260at this time, as this wash solution is not suitable for analysis by themass spectrometer.

The bottom portion of FIG. 2 shows the system in an elution and analysisconfiguration 200B. In this configuration, the wash solvent has flowedthrough the filtrate reservoir, taking the sample and standards with itthrough the chromatography portion 240 of the cartridge 205. The elutionsolvent is now flowing through the cartridge 205, assisted by a pump 210in the solvent assembly 201. The switch 225 directs the solutioncontaining elution solvent to the mass spectrometry portion of thesystem 215. The solution is atomized and the constituents ionized by anionization source in the MS part of the system. Sufficient fractionationshould occur during this process so that salts and buffers do notinterfere with the ionization or detection of the target analyte oranalytes. Following use, the user or system may dispose of thesingle-use cartridge. As mentioned above, single use of the cartridgeprevents cross contamination of the chromatography media.

The solvent assembly 201 shown in FIG. 2 includes sources for twosolvents, A and B, which can be the wash and elution solvents, as wellas a pump 210 which can selectively draw from the sources for solvents Aand/or B. The sources for solvents A and B are not shown, but they canbe reservoirs, such as flasks, beakers, bladders, and the like, whichcontain volumes of A and B. Alternately, or additionally, the pumps canbe syringe-type pumps that directly drive liquid flow from a reservoir.The volumes of A and B can be premixed, commercially availablesolutions, or the volumes of A and B can be prepared by a skilled userin order to tailor the solvents for use with the single-use cartridgeand the biological sample with the target analyte in mind.

The system shown in FIG. 2 can have one or more controllers that acceptsome user input to control the flow of solvent through the system andthe analytical instruments. One or more controllers can control the flowof solvent by controlling mechanisms such as the syringe pump 210 and/orthe switch 225. One or more controllers can also control theintroduction of liquid sample into the cartridge through the sampleintroduction port 215. The analysis of fractionated solvent, includingany ionization or other preparation that is done prior to massspectrometric analysis can be controlled by one or more controllers thatmay receive input from a user. The one or more controllers can includeuser interfaces, as well as computer processors, memory facilities, datatransmission facilities, and the like.

FIG. 3 shows a solvent assembly 300 that includes two syringes, inaccordance with some example embodiments. Each syringe includes aplunger assembly 305A and 305B, an outlet port 320A and 320B, solventreservoir 311A and 311B, as well as a seal 321A and 321B between thereservoir and the outlet port. The wash solvent 310 is contained withinthe wash reservoir 311A and the elution solvent 315 is contained withinthe elution reservoir 311B. Each plunger assembly 305A and 305B can beoperated separately by a syringe pump, such as pump 210 in the assembly201, or by a user by hand, separately to expel the wash solvent 310 andthe elution solvent 315.

The wash 310 and elution 315 solvents can mix in a single delivery lineor in a separate reservoir that fluidly connects to the single-usecartridge. For example, a first fluid delivery line connected to thewash outlet port 320A and a second line connected to elution outlet port320B can feed into a “Y” or “T” shaped junction that leads to a thirdline. In the third line, the wash and elution solvents can mix and thenflow into the single-use assay specific cartridge. Alternatively, afirst fluid delivery line connected to the wash outlet port 320A and asecond line connected to elution outlet port 320B can feed into a mixingreservoir. The wash and elution solvents can combine in the mixingreservoir and then flow toward the sample and chromatography portion ofthe single-use assay specific cartridge. The mixing reservoir can beexternal to the cartridge or part of the single-use assay specificcartridge.

Each plunger assembly 305 can be operated separately, as indicatedabove. Each plunger can be moved by a user's hand or by a motorizedmechanism, such as syringe pump 210. A syringe pump 210 can becontrolled by an instantaneous user's input, for example, by a push of abutton, selection of a value, or typing a command on a controller by auser. Alternatively, or additionally, a program can automaticallycontrol a syringe pump 210 to move each plunger assembly 305. Such aprogram can be selected by the system or user and can move one or bothof the plunger assemblies 305 a predetermined amount, in a series ofmotions, to dispense predetermined amounts of solvent.

The solvent assembly 300 can have two syringes and one or more keys,such as a unique shape at one of, or between, the outlet ports 320. Theone or more keys can correspond to one or more features on thesingle-use cartridge so that the solvent assembly can connect to thesingle-use cartridge in only one way. Limiting the connectionconfiguration of the solvent assembly and single-use cartridge can avoiderrors in solvent use.

For example, the outlet ports 320 can be shaped with a tapered end,similar to Luer-type fittings. In configurations where each of theoutlet ports 320 are similar, there can be bridge between the outletports 320 that is not straight, that is to say it is curved or angled,so that if the wash solvent plunger 305A is not inserted over the washsolvent inlet port on the cartridge, the solvent assembly 300 will notfit into the cartridge. Alternatively, or additionally, the one or moreof the outlet ports 320 can be asymmetrically shaped or have a notchedportion, so that the solvent assembly will not fit into the cartridge ifnot inserted correctly.

The seal of each syringe 321A and 321B can be a material that iscompatible with the solvent in each syringe, such that the seal will notdegrade over time when exposed to the solvent. The seal of each syringe321A and 321B can also be a material that is robust enough to endureshipping in various conditions, yet one that pierces without excessiveeffort when impaled on a pointed surface. Materials that can be used tofabricate the seal include polymers, such as polypropylene, latex,rubber, nitrile, ethylene tetrafluoroethylene (ETFE),polytetrafluoroethylene (PTFE), or any suitable material that iscompatible with the selected solvent. The seal 321 can be applied to theentire interior of the reservoir 311 of each syringe or only near, oron, the outlet port 320. The seal 321 can be applied duringmanufacturing of the syringes, such as just prior to filling the syringewith solvent or after filing the syringe with solvent. Additionally, thesyringe walls and each plunger assembly can be made of materialcompatible with the solvent contained within the syringe. Exemplarysyringe and plunger assembly materials can include polypropylene,polyolefins, polycarbonate, Teflon-type fluoroplastic resins (e.g.,PTFE), as well as metals, glass, ceramics, and other material able toresist degradation under exposure to the solvent, as well as theoperating pressures.

In some example embodiments, the single-use, assay specific cartridgecan directly accommodate the solvent assembly 300 shown in FIG. 3. Sucha single-use cartridge 405 is shown in FIG. 4. From the top view 400A ofthe single-use cartridge, solvent inlet ports 410, a solvent mixingchamber 425, a sample introduction port 215, a filter 220, achromatography portion 240 with immobilized chromatography media, and anoutlet port 460 can be seen. Safe-use features such as a bar code 445, anotched corner for directional insertion 450, and/or a key (e.g., aunique shape) 455 associated with at least one of the solvent inletports 410 to ensure that the solvents are inserted into the correct portare also shown in FIG. 4. The solvent inlet ports 410 can be specific interms of the type of solvent, wash or elution, that each accepts. Thespecificity of the inlet ports 410 can be dictated by one or more keys,such as each port having a different shape 455.

In the top view 400A, it can be seen that the outlet ports 320A and 320Bof the solvent assembly 300 connect directly to the solvent inlet ports410, such that each syringe of the solvent assembly connects to acorresponding inlet port. Wash solvent and elution solvent that entersthe cartridge 405 through the solvent inlet ports 410 flow into thesolvent mixing chamber 425. In the solvent mixing chamber 425, the washsolvent combines with the elution solvent. The incoming solvent isforced into the solvent mixing chamber 425 by a mechanism that actuatesthe plunger assemblies 305 of the solvent assembly 300, such as asyringe pump 210 or a mechanical actuator, like a stepper motor.Eventually, the mixed solvent flows into the filtrate reservoir 430.Before the mixed solvent flow into the reservoir 430, the filtratereservoir 430 can contain internal standards and filtered sample that issample without precipitates such as cells. The filtered sample combinedwith the mixed solvent flows into the chromatography portion 240 of thecartridge, flowing over the immobilized chromatography media. Thebiological sample is fractionated by the chromatography media in thechromatography portion 240 and passes through the outlet port 460 eitherto a waste receptacle 260 or to the mass spectrometric analysis portionof the system 235. In some example embodiments, a switch 225 directs theflow of fractionated solvent, such as by selectively opening valves orby repositioning conduits between the outlet and either the wastereceptacle 260 or the mass spectrometry system 215.

The side view of the cartridge 400B, allows for a better view of therelative location and size of the solvent inlets 410, the mixing chamber324, the filtrate reservoir 430, as well as the ports. The solventinlets ports 410, sample introduction port 215, and the outlet port 460are shown as situated on the top portion of the cartridge 405, connectedto chambers and reservoirs via fluid conduits. The solvent inlets 410are at a first end of the cartridge 405, opposite the end of thecartridge 405 with the outlet port 460. The sample and internal standardintroduction port 215 is located in the central portion of the cartridge405, directly over the filtrate reservoir 430.

In some example embodiments, the internal standards used with thesingle-use cartridge 405 are pre-loaded into the cartridge 405, such aswithin or on the filter 220, or within the filtrate reservoir 430.Alternatively, the user or system can introduce the internal standardsvia the sample introduction port 215. In some embodiments, thesingle-use assay specific cartridge 405 can operate with both pre-loadedinternal standards, as well as with internal standards materialsintroduced through the sample introduction port 215. Such flexibilitywith respect to the internal standard can increase the number of assaysa single-use cartridge can be used for.

Codes on the cartridge and solvent assemblies can be included to ensurecompatibility between the single-use, assay specific cartridge and thesolvent assemblies, including the solvent within the syringes of thesolvent assemblies. Codes can include machine readable codes, bar codes,RFID (radio frequency) codes, quick recognition (QR) codes, suitablevisual codes, suitable electromagnetic codes, and the like. The systemcan read the code on the cartridge prior to introduction of the sample,after introduction of the sample, before piercing the seals on thesolvent assemblies, or any combination thereof. The code on the solventassembly can be read by the system before piercing the seals on theassembly, before reading the code on the cartridge, or after reading thecode on the cartridge. The system can alert the user to anincompatibility between the cartridge and solvent assembly with a visualalarm, an audio alarm, or both. The system can also prevent an analysisfrom progressing when incompatible solvents are detected in the form ofan incompatible solvent assembly.

FIG. 4 shows a bar code 445 located on one corner of the top portion ofthe single-use cartridge 405, adjacent the outlet port 460. The notchedcorner 450 that acts as a key to prevent improper insertion of thecartridge 405 into a system is shown opposite the bar code 445, adjacentthe outlet port 460. The notched corner 450 is shown as continuingthrough the thickness of the cartridge 405, but in some exampleembodiments, a notched corner in a single-use cartridge can pass onlypartially through the thickness of the cartridge. Additionally, oralternatively, the bar code 445, or other codes that indicate which typeof assay the cartridge 405 is compatible with, can be located on otherportions of the cartridge. The other portions of the cartridge caninclude any portion that can be read by a user or the system, such as ona lateral side of the cartridge, on another corner of the top portion ofthe cartridge, on the back of the cartridge on the side opposed to allof the inlet ports, or any combination thereof.

FIG. 5 shows cross-section 500 of a portion of the single-use assayspecific cartridge 405 that cuts through one of the solvent inlets 410.The solvent inlet 410 shown includes a tapered fitting 511 that matchesthe shape of the outlet of one of the solvent syringes, as well as apiercing mechanism 513 that is configured to break the seal on thesyringe near the syringe tip or outlet. The piercing mechanism 513 canbe sharp, pointy, with at least one opening to a fluid conduit thatallows solvent to flow toward the solvent mixing chamber 425 in thesingle-use assay specific cartridge 405. In FIG. 5, the piercingmechanism 513 is shown as triangular components that protrude up fromthe surface of the cartridge 405, about the center of the solvent inlet410. The piercing mechanism 513 can include annular shapes, discretespikes or columns, as well as annular shapes with perforations throughtheir walls that lead to a conduit that allows solvent to flow towardthe solvent mixing chamber 425.

FIG. 6 shows a syringe of the solvent assembly of FIG. 3 interfacingwith a solvent inlet port 410, as shown in FIG. 5. The arrow within theassay solvent reservoir shows that piercing the seal 321 allows forsolvent flow from the syringe into the fluid conduit in the single-useassay specific cartridge 405 towards the solvent mixing chamber 425. Theseal 321 that separates the solvent within the syringe from theoutermost portion of the outlet port 320 is pierced by the piercingmechanism 513 when the syringe is fitted with the outlet port 320 of thesyringe in the tapered fitting 511, resulting in a pierced seal 622. Thetight fit between the outlet port 320 of the syringe and the taperedfitting 511 allows solvent to flow only into the cartridge 405. Thepiercing mechanism can break the seal 321 when the system is sealed orclosed, such as during the start of operation of the analysis system.

FIG. 7 shows an exemplary flow of solvent out of the single-usecartridge 405, through a conduit 702, from the chromatography media 240towards the mass spectrometry portion of the system 235. The outlet portof the cartridge 460 can have a tapered fitting that receives an adapter710 that connects to standardized tubing 715 that accommodates industrystandard HPLC tubing 720. The high-pressure liquid chromatography (HPLC)industry standard tubing 720 can connect to the conduit 702 from thechromatography media and convey solvent with analytes and internalstandards to the mass spectrometer portion of the system. The taperedfitting at the outlet port 460 can be a Luer-type fitting in which needsno piercing mechanism. In some embodiments, leak-tight fluidicconnections between the tapered fitting at the outlet port 460 and theadapter 710 can be established when the system is sealed, that isclosed-up, prior to starting analysis.

In some example embodiments, a single-use assay specific cartridge canbe supplied with a solvent assembly in an assay kit, such as in a sealedpouch. The kit can be designed for use with a specific instrument thatcan control the addition of a biological sample and the solvents. Theinstrument can also control atomization and ionization of the sample,and eventual analysis using a mass spectrometer.

In an implementation, a user inserts the cartridge in the orientationthat is accepted by the instrument. That orientation is dictated by thenotch in the cartridge shown in FIG. 4. Similarly, the solvent assemblyis inserted into the instrument in the orientation dictated by a keyfeature, as described above. Closing a lid on the instrument triggersthe system to verify the compatibility of the cartridge and the solventassembly, such as by verifying that the codes match, as well as fluidlyjoining the solvent assembly to the single-use cartridge oncecompatibility is confirmed.

The user adds at least a minimum required amount of biological sample,such as blood, through an opening in a lid of the instrument that allowsaccess to the sample inlet port on the cartridge. The biological samplesolubilizes the one or more dried internal standards within thecartridge. The sample moves through the filters so that cells,platelets, lipids, and other precipitates are removed from the sample,and the precipitate-free sample is collected in the filtrate reservoir.The filtrate reservoir has a metered volume, such as 3 microliters, thatis a known volume. The user or system fills the filtrated reservoir tothe known volume with biological sample before fractionation begins sothat concentrations and amounts of analyte can be calculated by thesystem. The user or instrument then blocks the sample inlet port, andthe instrument causes the solvents to flow through the cartridge, asdescribed above, fractionating the constituents in the sample so thatreliable results can be obtained from the mass spectrometer.

Though the system has been described in use with one single-use assayspecific cartridge, the system may be configured to accept multiplecartridges. For example, multiple single-use assay specific cartridgesmay be loaded into a system, each cartridge testing for differentanalytes, so that biological samples from a single patient can be testedserially, in an automated fashion. Similarly, methods that describeusing a system with only one single-use assay specific cartridge can beapplied to systems that can accept and utilize multiple assay specificcartridges at once.

Without in any way effecting the scope of the claims, a technical effectof the systems, methods, apparatus, and kits, described herein includestime and labor savings, elimination of potential errors, streamlinedlogistics and inventory management, improved safety, elimination ofsolvent quality control and quality assurance, and/or elimination ofsolvent waste.

The systems, methods, apparatus, and kits, described herein can be usedin many situations, including situations in which a patient arrives inan emergency room and is suspected to have, or exhibits, the followingconditions: acidosis, intentional overdose, uncontrolled seizures orconvulsions, being comatose or unresponsiveness, and cardiovasculararrhythmia and/or hypotension, usually seen in geriatric patients. Insome such instances, the analysis system does not need to be absolutelyaccurate in terms of the amount of analyte present in a sample, ratheronly the detection of a toxic or overdose level of an analyte needs tobe determined. The following are exemplary situations in which systems,methods, apparatus, and kits, described herein can find use.

Metabolic Acidosis: When a patient presents with acidosis (i.e.: lowblood pH) there are several possible causes. Ingestion of a toxicalcohol such as methanol or ethylene glycol leads to acidosis, but therecould be other causes not related to a toxic exposure such asstarvation, metabolic disorders, diabetes, asthma, strenuous exercise,and the like. There are few tools at the disposal of the physician(e.g., pH measurements, osmololal gap measurement) to make a diagnosisas to the cause of acidosis or to direct the course of treatment.

Toxic alcohol induced acidosis presents similarly to metabolic acidosis,but these conditions have very different treatments. Toxic alcoholexposure requires fomepizole treatment at low concentrations (e.g.,below 50 mg/mL) and hemodialysis at higher concentration (e.g., above 50mg/mL), while metabolic acidosis requires treating the underlyingcondition. As toxic alcohol poisoning can cause permanent damage (e.g.,optic nerve and kidney toxicity) and potentially be fatal if not treatedpromptly, patients with acidosis and suspected exposure are oftendialyzed as precautionary measure. The current assay for toxic alcoholsrequires specialized instrumentation (e.g., gas chromatography (GC) orGCMS) and trained staff that are not widely available.

The ideal assay panel would measure the concentration of toxic alcoholsand their metabolites along with several markers of metabolic activityin the patient. Lactate and pyruvate levels are key markers of theenergy state in a healthy cell. If there is a metabolic disorder, thelactate/pyruvate levels become altered, so these levels can provide animportant diagnostic clue to a physician or other caregiver.

Specific assays of interest in the toxic alcohol panel can includemethanol, isopropanol, ethylene glycol and their metabolites formicacid, acetone, and glycolic acid, respectively. Lactate and pyruvate areof interest to help in diagnosing causes of acidosis or ketosis that maybe unrelated to exposure to toxic alcohols. One kit could test apatient's blood or urine for these alcohols using the systems andmethods described herein, as well as possibly indicating the levels, orrelative levels, of lactate and pyruvate.

Intentional overdose (OD) panel: The archetypal OD patient is a 16-25year old female who intentionally overdoses on prescription medication.Often the drug, typically an antidepressant or anxiety medication, thatwas ingested by the patient is known to the emergency room physician.Wellbutrin and Celexa in particular need to be monitored very closelydue to possibility of late onset seizures, sometimes days after theinitial exposure. Furthermore, the current standard of care is to testeach and every intentional overdose patient for Tylenol (acetaminophen)and Aspirin (salicylic acid) given the ability of these common drugs tocause liver damage and that can result in complications for an ODpatient. A kit could include solvents and a cartridge includingchromatography media to fractionate samples so that acetaminophen,salicylic acid, as well as anti-depressants at toxic levels could bedetected. This could be a single kit or two or more kits.

Uncontrolled convulsions or seizures: When a patient is admitted to theER with uncontrolled seizures or convulsions there may be multiplecauses including an underlying seizure disorder like epilepsy or acompletely unrelated non-physiological cause such as an overdose of adrug such as cocaine or methamphetamines that cause convulsions. Theseizure panel would test the patient for commonly prescribedanti-convulsant drugs like Lamotrigine, carbamazepine and Keppra todetermine if the patient has a sub-therapeutic systemic dose due tomissed medications or a change in the presentation of the disease. Thepanel would also monitor compounds known to cause convulsions orseizures such as amphetamines, cocaine, buproprion (wellbutrin),citalopram (celexa) and possibly others. This panel could consist of twoassay kits—the first designed to prepare samples for detectingmedications to treat seizure disorders and the second designed toprepare samples for detecting likely toxin induced seizure agents.

While the panel would not be capable of looking at every drug known tocause or prevent seizures, a set of assay kits that cover the top 4-5agents would help physicians to diagnose a large percentage of patients.The treatment for a patient who has missed several doses of his seizuremedication is very different from one who has overdosed on cocaine, soknowing what has been ingested by a patient is crucial.

Coma Panel: Another common ER patient is an unresponsive or comatosepatient. In this case the physician is interested in determining whetheror not the patient has ingested a large amount of sedatives, includingprescription and illegal opiates, benzodiazepines, and heroin. Not everypossible drug or sedative can be measured in a single panel, but assaykits can focus on the most commonly observed agents. Even a negativeresult for the agents being assayed is important, as it will help thephysician rule out the most obvious and common causes and allow him orher to shift attention to other possible causes for the condition of thepatient.

Cardiovascular Panel: The archetypal patient is an elderly person witharrhythmia, bradycardia, and/or hypotension. Many of these patients areon multiple medications and it is common that dosing of one or more ofthe drugs is incorrect, either due to lack of patient compliance or achange in the underlying disease. An assay kit could be designed toallow the systems described herein to detect beta-blockers, calciumchannel inhibitors, and anti-arrhythmia drugs, specifically propafenone,clonidine, guanfacine, and the like. However, over time, the list ofparticular drugs can change as pharmaceutical markets evolve. A rapidblood test can help the physician diagnose the potential issue with thepatient, which is often due to an accidental overdose or missed doses(i.e.: an underdose) of an already prescribed medication.

The subject matter described herein may be embodied in systems,apparatus, methods, and/or articles depending on the desiredconfiguration. For example, the scanning system (or one or morecomponents therein) and/or the processes described herein can beimplemented using one or more of the following: a processor executingprogram code, an application-specific integrated circuit (ASIC), adigital signal processor (DSP), an embedded processor, a fieldprogrammable gate array (FPGA), and/or combinations thereof. Thesevarious implementations may include implementation in one or morecomputer programs that are executable and/or interpretable on aprogrammable system including at least one programmable processor, whichmay be special or general purpose, coupled to receive data andinstructions from, and to transmit data and instructions to, a storagesystem, at least one input device, and at least one output device. Thesecomputer programs (also known as programs, software, softwareapplications, applications, components, program code, or code) includemachine instructions for a programmable processor, and may beimplemented in a high-level procedural and/or object-orientedprogramming language, and/or in assembly/machine language. As usedherein, the phrase “machine-readable medium” refers to any computerprogram product, computer-readable medium, apparatus and/or device(e.g., magnetic discs, optical disks, memory, Programmable Logic Devices(PLDs)) used to provide machine instructions and/or data to aprogrammable processor, including a machine-readable medium thatreceives machine instructions. Similarly, systems are also describedherein that may include a processor and a memory coupled to theprocessor. The memory may include one or more programs that cause theprocessor to perform one or more of the operations described herein.

Although a few variations have been described in detail above, othermodifications or additions are possible. In particular, further featuresand/or variations may be provided in addition to those set forth herein.For example, the implementations described above may be directed tovarious combinations and subcombinations of the disclosed featuresand/or combinations and subcombinations of several further featuresdisclosed above. In addition, the logic flow depicted in theaccompanying figures and/or described herein does not require theparticular order shown, or sequential order, to achieve desirableresults. In various example implementations, the methods (or processes)can be accomplished on mobile station/mobile device side or on theserver side or in any shared way between server and userequipment/mobile device with actions being performed on both sides. Thephrases “based on” and “based on at least” are used interchangeablyherein. Other implementations may be within the scope of the followingclaims.

What is claimed:
 1. A system for determining the concentration of ananalyte of interest in a biological sample, the system comprising: asolvent assembly comprising a first volume of a first solvent and asecond volume of a second solvent, the solvent assembly furthercomprising a first outlet fluidly connected to the first volume and asecond outlet fluidly connected to the second volume; and a disposablecartridge comprising: a solvent inlet configured to introduce at leastone of the first and second solvents from the solvent assembly into thedisposable cartridge, wherein the solvent inlet comprises a firstsolvent introduction port and a second solvent introduction port, thefirst solvent introduction port configured to accept the first outlet ofthe solvent assembly and to fluidly connect to the first volume, thesecond solvent introduction port configured to accept the second outletof the solvent assembly and to fluidly connect to the second volume, orwherein the solvent inlet comprises a single solvent introduction portconfigured to fluidly connect to both the first outlet and the secondoutlet via a joining portion between the first and second outlets; asample introduction port configured to receive the biological sample,the sample introduction port fluidly connected to the solvent inlet; achromatography region fluidly connected to the sample introduction port;and an outlet fluidly connected to the chromatography region, andwherein to provide an indication that the solvent assembly and thedisposable cartridge are compatible for performing an assay to determinethe concentration of the analyte of interest, at least a portion of thesolvent assembly has an asymmetrical shape that fits together in aspecific orientation to at least a portion of the solvent inlet having ashape complementary to the asymmetrical shape.
 2. The system of claim 1,further comprising a sample analysis portion operably connected to theoutlet of the disposable cartridge, wherein the sample analysis portioncomprises a mass spectrometric analysis system, wherein the massspectrometric analysis system produces a spectra for the analyte ofinterest, and produces at least one of a plurality of isotopes of theanalyte of interest or a plurality of derivatized samples of the analyteof interest added to the biological sample in a single spectralmeasurement.
 3. The system of claim 1, further comprising at least oneprocessor having operational thereon computer instructions for at leastcontrolling operation of the system, to at least determine aconcentration of the analyte contained in the biological sample and toreport the at least one analyte concentration.
 4. The system of claim 1,further comprising immobilized chromatography media in thechromatography region of the disposable cartridge.
 5. The system ofclaim 4, wherein the immobilized chromatography media corresponds to oneor more predetermined assays.
 6. The system of claim 1, furthercomprising one or more filters in the disposable cartridge, wherein theone or more filters are configured to remove precipitates and/orinsoluble material from the biological sample and yield a filteredbiological sample.
 7. The system of claim 6, further comprising afiltrate reservoir in the disposable cartridge, wherein the filtratereservoir has a metered volume and is configured to contain the filteredbiological sample.
 8. The system of claim 6, further comprising one ormore internal standards, wherein the one or more internal standards areprovided in a dried form.
 9. The system of claim 8, wherein the one ormore internal standards correspond to one or more predetermined assays.10. The system of claim 1, wherein the solvent assembly and thedisposable cartridge are constrained, by at least the asymmetrical shapeof the portion of the solvent assembly, to fit together in the specificorientation.
 11. The system of claim 10, wherein to provide theindication that the solvent assembly and the disposable cartridge arecompatible, the first outlet has the asymmetrical shape and the firstsolvent introduction port has the shape complementary to theasymmetrical shape of the first outlet, or the second outlet of thesolvent assembly has the asymmetrical shape and the second solventintroduction port has the shape complementary to the asymmetrical shapeof the second outlet, or both the first and second outlets have theasymmetrical shape and the first and second solvent introduction portshave the shape complementary to the asymmetrical shape of the first andsecond outlets, or the joining portion between the first and secondoutlets has the asymmetrical shape and the single solvent introductionport has the shape complementary to the asymmetrical shape of thejoining portion.
 12. The system of claim 11, wherein the disposablecartridge further comprises a receiver structure and wherein thereceiver structure comprises at least one of the first solventintroduction portion or the second solvent introduction port of thedisposable cartridge.
 13. The system of claim 1, wherein the assaycomprises at least one of a metabolic acidosis assay, an intentionaloverdose assay, an uncontrolled convulsions or seizures assay, a comaassay, or a cardiovascular assay.
 14. The system of claim 13, whereinthe assay comprises one or more test panels.
 15. The system of claim 1,further comprising one or more codes indicating the compatibility of thesolvent assembly with the disposable cartridge to perform the assay todetermine the presence of the analyte of interest.
 16. The system ofclaim 15, wherein the one or more codes comprise color coding, writtencodes, machine readable codes, electrical signaling codes, or anycombination thereof.
 17. The system of claim 16, wherein the writtencodes comprise alpha-numeric codes, symbols, or a combination of both.18. The system of claim 16, wherein the machine readable codes comprisesymbols, quick response (QR) codes, barcodes, or any combinationthereof.